Abstract Safety culture is a product of values, attitudes, skills, and behavioral patterns, and it determines the commitment of the management to a secure organization. The evaluation of safety culture in hospitals helps to identify and manage the relevant patient safety issues in hospital routines and working conditions proactively. Thus, this study is aimed to evaluate patient safety culture in all the departments of a university hospital of medium complexity. This study employed a cross-sectional and analytical design employed in the Portuguese version of the Hospital Survey on Patient Safety Culture. A sample of all hospital staff participated in this study, which was conducted from December 2016 to May 2017. The percentage of positive responses was calculated to identify the strong and weak areas in patient safety. Of the 413 questionnaires distributed, 368 valid responses were returned. The response rate was, therefore, 89%. The overall percentage of positive responses was 50.3%. The "Supervisor/manager expectations and actions promoting patient safety" dimension obtained the highest percentage of positive responses (67.1%). The "Nonpunitive response to error" dimension was considered the weakest for safety culture, with only 22.9% positive responses. Furthermore, most professionals (70.6%) did not report any events in the previous 12 months. Nevertheless, 69.5% of participants considered patient safety within their unit/work area as "very good" or "great." The results showed that the employees' perception of patient safety diverged from the reality within the institution. Therefore, efforts should be made to promote an acceptable safety culture in all hospital areas.